Template fixed β-hairpin loop mimetics and their use in phage display

ABSTRACT

Template-fixed β-hairpin mimetics and libraries including a plurality of these mimetics are provided. The template-fixed β-hairpin mimetics are of the following general formula:
 
R 1 -Cys-Z-Cys-R 2   I
 
wherein the two cysteine residues are bridged by a disulfide bond, thereby forming a cyclic peptide; R 1  and R 2  are di- or tripeptide moieties of certain types, as defined herein; and Z is a chain of n amino acid residues with n being an integer from 4 to 20 and with each of these n amino acid residues being, independently, derived from any naturally occurring L-α-amino acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/579,104, filed May 12, 2006, and afforded a date of receipt of 35U.S.C. 371(c)(1), (c)(2), and (c)(4) requirements of Nov. 2, 2006, whichU.S. application Ser. No. 10/579,104 is the national phase in the U.S.of International Application No. PCT/EP2003/012783, filed Nov. 15, 2003,the entire contents of all of which are incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention relates to compositions and methods of certainpeptide sequences consisting of residues of naturally occurringL-α-amino acids wherein certain amino acid residues, depending on theirpositions in the chains, are cysteines which are bridged by a disulfidebond, thereby forming cyclic peptides, and certain other amino acidresidues which are adjacent to the said cysteines form di- or tripeptidemoieties of certain types, as defined herein below, which together actas templates in order to facilitate the formation and stabilization ofβ-hairpin loop structures. By virtue of their stability and constraintsthese template fixed hairpin loop mimetics can exhibit higher orprolonged activity against protein binding partners.

The templates can be transplanted into the construction of phage displayderived hairpin loop mimetics for library screening and drug screening.Methods and compositions of the present invention are useful forscreening and identifying interacting proteins in vitro. The presentinvention can serve as an additional efficient lead finding tool fortargets where it is difficult to transfer protein epitopes from intosmall peptides or peptide mimetics.

BACKGROUND OF THE INVENTION

The surface loops of proteins and bioactive peptides have often beenimplicated in recognition by protein binding partners. Accordingly, itis of interest to investigate these loops as potential leads for drugdiscovery. Particularly of interest are β-hairpins: The β-hairpin motifis very abundant in nature and occurs on the surface of many proteinligands and in the hypervariable domains of antibodies. The β-hairpinmotif consists of two antiparallel β-strands linked by a short loop orturn and have been classified depending on the H-bonding network[Sibanda, B. L.; Blundell, T. L.; Thornton, J. M. J. Mol. Biol. 1989,206, 759-777].

The ability to generate β-hairpin peptidomimetics using combinatorialand parallel synthesis methods has now been established (L. Jiang, K.Moehle, B. Dhanapal, D. Obrecht, J. A. Robinson, Helv. Chim. Acta. 2000,83, 3097-3112). However these molecules may not be synthesized inlibraries as large as 10¹⁰ or 10¹².

A complementary strategy for peptide-based lead discovery consists ofdisplay of libraries on filamentous bacteriophage which allows thepreparation of libraries as large as 10¹⁰-10¹², many magnitudes largerthan libraries that may be prepared synthetically.

Furthermore rapid and inexpensive selection protocols are available foridentifying those library members that bind to a target of interest.Phage display technique allows the construction of cyclic constrainedpeptides such as disulfide-constrained β-hairpin loops, as is well known(H. B. Lowman, Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 401-24).

These loops represent a limited number of conformations which may resultin isolation of affinity ligands for a receptor target. Cyclic peptides,however, stabilized by only one disulfide bond are stillconformationally quite flexible. Also, it is well known that disulfidebond formation and cleavage can be reversible and flexibility isincreased by the fact that the peptide constraints are fused to theamino terminus of the gene III protein. Thus it is important tostabilize such loop constructs by additional residues, adjacent to thedisulfide bond which favor the β-sheet conformation. (R. H. Hoess,Current opinion in Structural Biology 1993, 3, 572-579). This may notlead to high affinity ligands for a receptor target. The same peptideloop is fixed in the natural protein scaffold by the protein scaffold onto N- and C-terminus of the loop and is additionally constrained byhydrogen bonds of anti-parallel beta-sheets which is induced by thenatural protein scaffold. Other approaches have been proposed such aspeptide scaffolds for turn display (A. G. Cochran, R. T. Tong, M. A.Starvasnik, E. J. Park, R. S. McDowell, J. E. Theaker, N. J. Skeleton,J. Am. Chem. Soc. 2001, 123, 625-632). Another possible solution to thisproblem is to use structural constraints of a folded protein to presentsmall variable peptide segments (P.-A. Nygren, M. Uhlen, Curr. Opin.Struc. Biol. 1997, 7, 463-469; G. P. Smith, S. U. Patel, J. D. Windass,J. M. Thornton, G. Winter, A. D. Griffiths, J. Mol. Biol. 1998, 277,317-332; A. Christmannn, K. Walter, A. Wenzel, R. Krazner, R. Kolmar,Protein Eng. 1999, 12, 797-806).

In fact the epitope transfer from proteins into small peptides remains aproblem (A. G. Cochran Chem. Biol. 2000, 7, R85-R94).

SUMMARY OF THE INVENTION

The invention described below provides peptide templates consisting ofresidues of naturally occurring L-α-amino acids, whose function is torestrain the peptide loop backbone into a hairpin geometry in astabilized β-hairpin conformation. These templates can be used for theconstruction of phage display derived template fixed β-hairpin loopmimetics generating phage display libraries with very high bindingconstants to targets.

This method provided by the invention can be advantageously used inscreening of large libraries of phage display derived template fixedβ-hairpin loop mimetics which in turn considerably facilitatesstructure-activity studies, and hence the discovery of new moleculeswith potent activities and with novel selectivities towards differenttypes of targets.

Due to the structurally and conformationally well-defined architectureof the β-hairpin loop mimetics of the general formula I, as definedhereinbelow, key amino acid residues or motifs within the chainZ—encoded as nucleic acid sequences in phage display libraries—can beintegrated in conformationally locked arrangements. By shifting thesekey amino acid residues or motifs along the β-hairpin structure, newarrangements of important amino acids can be scanned (positionalscanning of key sequences). Alternatively, protein sequences can bemapped in order to detect β-hairpin loop motifs. This technique, insummary, allows determining rapidly key amino acids and motifs(hotspots) important for binding in large surface and flat proteininterfaces not only in their sequential but also in their spatialarrangement. This information can ultimately be used for the design ofsmall peptidomimetic drug candidates (Cunningham, B. C.; Wells, J. A.Curr. Opin. Struct. Biol. 1997, 7, 457; Obrecht, D.; Altorfer, M.;Robinson, J. A. Adv. Med. Chem. Vol. 4, 1-68, JAI Press Inc., 1999).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-6 show a comparison of the formation of disulfide bridgedβ-hairpin mimetics in % up to a time after which no progress ofconversion was detected.

FIG. 7 shows a CD spectra (ξ deg×cm²/mol) of the linear peptideprecursors of the compounds of Examples 3, 4, 5 and 9, i.e. beforedisulfide bridge formation.

DETAILED DESCRIPTION OF THE INVENTION

The template fixed β hairpin loop mimetics of the present invention arecompounds of the general formulaR¹-Cys-Z-Cys-R²  Iwhereinthe two Cys residues are bridged by a disulfide bond thereby forming acyclic peptide;R¹ and R² areA-B and B-C; or B-A and C-B; or C-B and B-A; or B-C and A-B; or C-A andC-A; or A-C and A-C; or C-A and C-B; or B-B and C-B; or B-B and B-C; orA-B and C-C; or B-A and C-C; or C-B and B-B; or B-C and B-B; or C-C andB-A; or C-C and A-B; or B-B and C-C; or C-C and B-B; or A-C and B-C; orC-B and C-A; or B-C and A-C; or A-C and A-B; or B-A and C-A; A-A andC-C; or C-C and A-A;or A-B-C and A-B-C; or B-A-B and B-C-B; or B-C-B and B-A-B; or A-B-B andB-B-C; or C-B-B and B-B-A; or A-C-B and B-A-C; or C-A-B and B-C-A; orB-A-B and B-C-C; or B-C-B and B-A-C; or C-C-B and B-B-A; or C-C-B andB-A-B, or C-B-B and C-C-A; or A-C-C and B-B-C; or B-C-C and B-A-B; orB-C-C and B-A-C; or A-B-B and B-C-C; or B-A-B and C-C-B; or C-A-B andC-C-B; or B-B-B and B-C-C; or C-B-B and B-B-B; or B-B-B and C-C-B; orB-C-C and B-B-B; or A-B-C and B-B-C; or C-B-B and C-B-A; or A-B-C andA-C-C; or C-C-A and C-B-A; or B-A-C and A-C-B; or B-C-A and C-A-B; orC-B-A and C-B-A; or A-A-B and B-C-C; or C-C-B and B-A-A; or B-B-C andA-C-C; or B-B-C and A-B-C; or B-B-C and B-B-C; or B-B-C and B-B-B; orB-A-C and B-C-C; or C-C-B and C-A-B; or C-C-B and C-B-A; or A-B-C andB-C-C; or C-A-B and B-C-B; or B-C-B and B-B-C; or C-B-B and B-C-B; orB-C-B and B-B-B; or B-B-B and B-C-B; or C-B-B and B-C-A; or A-C-B andB-B-C; or C-B-B and C-B-B; or B-B-B and B-B-B; or B-B-B and B-B-C; orA-A-C and A-C-C; or C-C-A and C-A-A; or A-A-C and A-C-B; or B-C-A andC-A-A; or A-A-C and B-C-C; or C-C-B and C-A-A; or A-A-B and C-C-B; orB-C-C and B-A-A; or A-B-A and C-B-C; or C-B-C and A-B-A; or A-B-B andC-B-C; or C-B-C and B-B-A; or B-A-A and C-C-B; or B-C-C and A-A-B; orB-B-A and C-B-B; or B-B-C and A-B-B; or B-B-A and C-C-B; or B-C-C andA-B-B; or B-B-C and A-C-B; or B-C-A and C-B-B; or B-C-B and C-B-B; orB-B-C and B-C-B; or B-C-B and C-A-B; or B-A-C and B-C-B; or B-C-B andC-B-B; or B-A-C and A-C-B; or B-A-C and A-C-C; or C-C-A and C-A-B; orB-A-C and B-C-C; B-C-C and A-A-C; or C-A-A and C-C-B; or C-A-A andC-C-A; or A-C-C and A-A-C; or C-B-A and C-C-A; or A-C-C and A-B-C; orC-B-A and C-B-B; or C-B-A and C-C-B; or B-C-C and A-B-C; or C-B-B andC-C-A; or C-B-B and C-B-B; or C-B-B and C-C-B; or B-CC and B-B-C; orC-C-A and C-A-B; or C-C-A and C-B-B; or C-C-B and B-B-B; or C-C-B andC-A-A; or C-C-B and C-B-A; or C-C-B and C-B-B;or B-B-C and B-C-C; or A-C-B and B-B-C; or A-C-C and B-B-C;A being any one of Asn, Gln, Asp, Glu, Thr, Ser and Gly;B being any one of Val, Ile, Ser, Thr, Phe, Tyr, Trp and Gly; andC being any one of Arg, Lys and Gly; andZ is a chain of n amino acid residues with n being an integer from 4 to20 and with each of these n amino acid residues being, independently,derived from any naturally occurring L-α-amino acid.

For example, Z contains one of the key sequences -Arg-Gly-Asp-,-Glu-Leu-Arg-, -Arg-Lys-Lys- and -Lys-Gly-Phe- or consists of, orcontains one of the key sequences -Val-Arg-Lys-Lys- [SEQ ID NO:1],-Lys-Lys-Tyr-Leu- [SEQ ID NO:2], -Trp-Leu-Asp-Val- [SEQ ID NO:3],-Tyr-Ile-Arg-Leu-Pro- [SEQ ID NO:4], -Tyr-Ile-Gly-Ser-Arg- [SEQ IDNO:5], -Ile-Lys-Val-Ala-Val- [SEQ ID NO:6], -Pro-Pro-Xaa-Xaa-Trp- [SEQID NO:7] wherein Xaa can be residues of any naturally occurringL-α-amino acids, -Leu-Trp-Tyr-Ser-Asn-His-Trp-Val- [SEQ ID NO:22],-Lys-Trp-Phe-Ser-Asn-His-Tyr-Gln- [SEQ ID NO:23],-Phe-Leu-Ala-His-Tyr-Ala- [SEQ ID NO:24] and-Leu-Trp-Tyr-Ser-Asn-His-Trp-Val-Lys-Trp- [SEQ ID NO:25]; these keysequences will be discussed in more detail hereinafter.

The library of template-fixed β-hairpin mimetics of the presentinvention comprises a plurality of compounds of the above generalformula I. This library of the template fixed β-hairpin mimetics can befused to at least a portion of phage coat protein, and the templatefixed β-hairpin mimetics are displayed on the surface of a phage orphagemid particle.

The invention also provides a screening method for template fixedhairpin β-mimetics having a template that conformationally stabilizes aβ-hairpin conformation and which are capable of binding to a specificbinding partner comprises the steps of

a) providing a library of template fixed β-hairpin mimetics of formula Iwhich may be fused to at least a portion of phage coat protein where thetemplate-fixed β-hairpin mimetics are displayed on the surface of aphage or phagemid particle;

b) contacting the library of step a) with a binding partner;

c) selecting from the library phage peptides capable of forming anon-covalent complex with the binding partner; and

d) optionally isolating the peptides or determining the sequence byDNA-analysis of step c).

In such method the binding partner is normally an antibody, an enzyme, areceptor or a ligand or fragments or portions thereof.

Phage peptides which have been determined and optionally isolated by theabove process and synthetic peptides having structures which areidentical to the structures of the peptides thus determined andoptionally isolated also form part of the present invention.

The structural elements forming the templates consist of the twodisulfide-bridged Cys residues together with the residues R¹ and R²,which comprise either both two or both three amino acid residues which,as described below, are capable of stabilizing β-sheet conformation andwhich are positioned on opposite sites of the antiparallel β-strandsadjacent to the disulfide bond; furthermore these templates have anN-terminus and a C-terminus oriented to be linked to the chain Z. Thepeptide chain Z is linked to the C-terminus and the N-terminus of thetemplates via the corresponding N- and, respectively, C-termini so thatthe template and the chain combine to a cyclic structure.

As amino acid residues there come into consideration those which arederived from naturally occurring L-α-amino acids. Hereinafter there isgiven a list of these amino acids which, or the residues of which, aresuitable for the purposes of the present invention, the abbreviationscorresponding to generally adopted usual practice.

-   -   Ala A L-Alanine    -   Arg R L-Arginine    -   Asn N L-Asparagine    -   Asp D L-Aspartic acid    -   Cys C L-Cysteine    -   Glu E L-Glutamic acid    -   Gln Q L-Glutamine    -   Gly G Glycine    -   His H L-Histidine    -   Ile I L-Isoleucine    -   Leu L L-Leucine    -   Lys K L-Lysine    -   Met M L-Methionine    -   Phe F L-Phenylalanine    -   Pro P L-Proline    -   Ser S L-Serine    -   Thr T L-Threonine    -   Trp W L-Tryptophan    -   Tyr Y L-Tyrosine    -   Val V L-Valine    -   R¹ and R² comprise either each two or each three amino acid        residues which hereinabove have been represented by the Symbols        A, B and C, each of which stands for one of the following        groups:    -   Group A: amino acid residues capable of formation of ion bond or        hydrogen bond interaction;    -   Group B: amino acid residues capable of formation of hydrophobic        interaction; and    -   Group C: amino acid residues capable of formation of cationic-π        interaction or ion bond or hydrogen bond.

Group A comprises amino acids containing side chains withpolar-non-charged or acidic residues. A polar-non-charged residue refersto a hydrophilic side chain that is uncharged at physiological pH. Suchside chains typically contain hydrogen bond donor groups such as primaryand secondary amides or alcohols. An acidic residue refers to ahydrophilic side chain that contains a carboxylic group. The naturallyoccurring polar-non-charged or acidic L-α-amino acids are asparagine,glutamine, aspartatic acid, glutamic acid, threonine, and serine.Glycine is included in group A as a neutral β-sheet former. The aminoacid side chains can form an interstrand ionic bond (salt bridge) or ahydrogen bond interaction with amino acid residues group C at oppositepositions of the anti-parallel β-sheet and, in addition, an intrastrandhydrogen or ionic bond interaction with amino acid residues of group Cof tripeptide moieties (Ciani B. et al., J. Am. Chem. Soc. 2003, 125,9038-9047; Searle, M. S. et al., J. Am. Chem. Soc. 1999, 121,11615-11620) within the template.

Group B comprises amino acid residues containing small to medium sizedhydrophobic, or aromatic or heteroaromatic, or polar-non-charged sidechain residues. A hydrophobic small- to medium-sized residue refers toan amino acid side chain that is uncharged at physiological pH. Anaromatic amino acid residue refers to a hydrophobic amino acid having aside chain containing at least one ring having a conjugated π-electronsystem (aromatic group). In addition they may contain hydrogen bonddonor groups such as primary and secondary amines and alcohols. Apolar-non-charged residue refers to a hydrophilic side chain that isuncharged at physiological pH. Such side chains typically containhydrogen bond donor groups such as alcohols. The naturally occurringsmall-to-medium-sized L-α-amino acids, aromatic and heteroaromaticL-α-amino acids and polar-non-charged L-α-amino acids are valine,isoleucine, serine, threonine, phenylalanine, tyrosine, and tryptophane.Glycine is included in group B as a neutral β-sheet former. The aminoacid side chains can form an interstrand hydrophobic-hydrophobicinteraction with amino acid residues group B or hydrophobic (π)-cationicinteraction with amino acid residues group C at opposite positions ofthe anti parallel β-sheet and, in addition, an intrastrand hydrophobicor hydrophobic-cationic interaction with amino acid residues of group Bor C of tripeptide moieties within the template.

Group C comprises amino acids containing side chains with polar-cationicresidues. Polar cationic refers to a basic side chain which isprotonated at physiological pH. The naturally occurring polar-cationicL-α-amino acids are arginine and lysine (Ciani B. et al., J. Am. Chem.Soc. 2003, 125, 9038-9047; Searle, M. S. et al., J. Am. Chem. Soc. 1999,121, 11615-11620). Glycine is included in group C as a neutral β-sheetformer. The amino acid side chains of arginine and lysine can form aninterstrand cationic-hydrophobic (π)-interaction (J. P. Gallivan, D. A.Dougherty, Proc. Natl. Acad. Sci. USA 1999, 96, 9459-9464) with aminoacid residues type B or ionic bond interaction (salt bridge) or hydrogenbond interaction with group A amino acid residues at opposite positionsof the anti parallel β-sheet and, in addition, an intrastrandcationic-hydrophobic (π)-interaction with amino acid residues of group Bof tripeptide moieties within the template.

R¹ and R² are preferably

-   -   Glu-Thr and Thr-Lys; or Lys-Thr and Thr-Glu; or    -   Thr-Glu and Lys-Thr; or Thr-Lys and Glu-Thr; or    -   Leu-Glu and Lys-Val; or Val-Lys and Glu-Leu; or    -   Glu-Leu and Val-Lys; or Lys-Leu and Val-Glu; or    -   Asn-Gly and Lys-Val; or Val-Gly and Lys-Asn; or    -   Gly-Asn and Val-Lys; or Gly-Val and Asn-Lys; or    -   Gly-Gly and Gly-Gly; or    -   Glu-Leu-Lys and Glu-Val-Lys; or Lys-Val-Glu and Lys-Leu-Glu; or    -   Leu-Glu-Lys and Glu-Lys-Val; or Val-Lys-Glu and Lys-Glu-Leu- or    -   Glu-Lys-Leu and Val-Glu-Lys; or Lys-Glu-Val and Leu-Lys-Glu; or    -   Lys-Glu-Leu and Val-Lys-Glu; or Glu-Lys-Val and Leu-Glu-Lys; or    -   Lys-Val-Gly and Gly-Leu-Glu; or Glu-Leu-Gly and Gly-Val-Lys; or    -   Val-Lys-Gly and Gly-Glu-Leu; or Leu-Glu-Gly and Gly-Lys-Val; or    -   Val-Gly-Lys and Glu-Gly-Leu; or Leu-Gly-Glu and Lys-Gly-Val; or    -   Gly-Gly-Gly and Gly-Gly-Gly.

The positions P¹ to P^(n) of each amino acid residue in the chain Z isunequivocally defined as follows: P¹ represents the first amino acid inthe chain Z that is coupled with its N-terminus to the C-terminus of thetemplate and P^(n) represents the last amino acid in the chain Z, thatis coupled with its C-terminus to the N-terminus of the template.

Advantageously the chain Z consist of, or contains, a key sequence oftwo, three, four, five, six or occasionally up to ten amino acidresidues, the two terminal members of which are “constant” (“k”) whilstany other members are either “constant”, too, or “variable” (“x”), inall possible combinations or permutations. The two terminal “constant”members can be the same or different, and the same applies to anyremaining “constant” and/or to any “variable” members.

The key sequences can be translated into oligo-nucleic acid sequencesand transplanted into phage displayed peptides of the invention.

Particularly suitable “constant” members (“k”) are Trp, Arg, Tyr, Ile,Asp, His, Lys, Glu and Thr, further suitable “constant” members (“k”)are Gln, Phe, Met and Ser, and suitable “variable” members (“x”) areAla, Leu and Val.

Key sequences of two, three, four, five and six amino acid residues, canbe schematically depicted as follows:

dipeptide -k¹-k²- tripeptide -k¹-k²-k³- -k¹-x¹-k²- tetrapeptide-k¹-k²-k³-k⁴- -k¹-x¹-k²-k³- -k¹-k²-x¹-k³- -k¹-x¹-x²-k²- pentapeptide-k¹-k²-k³-k⁴-k⁵- -k¹-x¹-k²-k³-k⁴- -k¹-k²-x¹-k³-k⁴- -k¹-k²-k³-x¹-k⁴--k¹-x¹-x²-k²-k³- -k¹-k²-x¹-x²-k³- -k¹-x¹-k²-x²-k³- -k¹-x¹-x²-x³-k²-hexapeptide -k¹-k²-k³-k⁴-k⁵-k⁶- -k¹-x¹-k²-k³-k⁴-k⁵- -k¹-k²-x¹-k³-k⁴-k⁵--k¹-k²-k³-x¹-k⁴-k⁵- -k¹-k²-k³-k⁴-x¹-k⁵- -k¹-x¹-x²-k²-k³-k⁴--k¹-k²-x¹-x²-k³-k⁴- -k¹-k²-k³-x¹-x²-k⁴- -k¹-x¹-k²-x²-k³-k⁴--k¹-k²-x¹-k³-x²-k⁴- -k¹-x¹-k²-k³-x²-k⁴- -k¹-x¹-x²-x³-k²-k³--k¹-k²-x¹-x²-x³-k³- -k¹-x¹-k²-x²-x³-k³- -k¹-x¹-x²-k²-x³-k³--k¹-x¹-x²-x³-x⁴-k²-

Certain key sequences are known to occur in important physiologicallyactive peptides, such as

R G D in fibronectin (FN), vitronectin  (VN), osteopontin, collagens, thrombospondin, fibrinogen (Fg),  von Willebrand factor (vWF), see Obrecht, D.; Altorfer, M.;  Robinson, J. A. Adv. Med. Chem. Vol. 4, 1-68, JAI Press Inc., 1999 E L Rin C X C chemokines, see Saunders,  J.; Tarby, C. M. Drug Discovery Today, 1999, 4, 80-92 R K K see J. Biol. Chem. 1999, 274, 3513 K G Fsee Prot. Sci. 1998, 7, 1681-1690 V R K K in Platelet-Derived Growth Factor  [SEQ ID NO: 1](PDGF), see Ross, R.; Raines,  E. W.; Bowden-Pope, D. F. Cell, 1986, 46, 155-159 K K Y L  in VIP (vasointestinal peptide) [SEQ ID NO: 2] showing neuroprotective properties against β-amyloid neurotoxicity,  see Proc. Natl. Am. Soc. USA 1999, 96, 4143-4148 W L D V  in integrin α₄β₁, see Europ. J. [SEQ ID NO: 3 Biol. 1996, 242, 352-362 and Int. J. Pept. Prot. Res. 1996, 47,  427-436 Y I R L P in Factor Xa inhibitors, see Al  [SEQ ID NO: 4]Obeidis,F.; Ostrem, J. A. Drug  Discovery Today 1998 , 3, 223-231Y I G S R  in laminine, see EMBO. J. 1984,  [SEQ ID NO: 5] 3, 1463I K V A V  see Cell 1987, 88, 989 [SEQ ID NO: 6] P P R X X W see J. Biol. Chem. 1998, 273,  [SEQ ID NO: 7] 11001-11006 & 11007-11011

Phage display is a technique by which variant polypeptides are displayedas fusion proteins to a coat protein on the surface of a phage,filamentous phage particles, as described in “Phage Display of Peptidesand Proteins”, B. K. Kay, J. Winter, J. Mc Cafferty 1996, AcademicPress.

As used in this description, the term “coat protein” means a protein atleast a portion of which is present on the surface of the virusparticle. The coat protein may be the major coat protein or may be aminor coat protein.

The term “electroporation” means a process in which foreign matter(protein, nucleic acid, etc) is introduced into a cell by applying avoltage to the cell under conditions sufficient to allow uptake of theforeign matter into the cell. The foreign matter is typically DNA.

A “fusion protein” is a polypeptide having two portions covalentlylinked together, where each of the portions is a polypeptide having adifferent property.

A “phagemid” is a plasmid vector having a bacterial origin ofreplication, ColE1, and a copy of an intergenic region of abacteriophage. The phagemid may be based on any known bacteriophage,including filamentous bacteriophage. Segments of DNA cloned into thesevectors can be propagated as plasmids. When cells harboring thesevectors are provided with all genes necessary for the production ofphage particles, the mode of replication of the plasmid changes torolling circle replication to generate copies of one strand of theplasmid DNA and package phage particles. The phagemid may forminfectious or non-infectious phage particles.

The term “phage vector” means a double stranded replicative form of abacteriophage containing a heterologous gene and capable of replication.The phage vector has a phage origin of replication allowing phagereplication and phage particle formation. The phage is preferably afilamentous bacteriophage, such as an M13 phage or a derivative thereof,a lambdoid phage such as, but not limited to, phi80, phages 21, 82, 424,432, lambda.imm343, lambda.imm21, lambda.EMBL or lamdab.gt., or allderivatives, genetically engineered derivatives, and hybrids thereof.

“Ligation” is a process of forming phosphodiester bonds between twonucleic acid fragments. For ligation of the two fragments, the ends ofthe DNA fragments need to be compatible with each other. In most cases,the ends will be directly compatible after endonuclease digestion.However, it may be necessary first to convert the staggered endscommonly produced after endonuclease digestion to blunt ends to makethem compatible for ligation. For the creation of blunt ends,DNA-modifying enzymes like T4 polymerase or Klenow are used under theconditions as described by the supplier.

DNA purification is performed by phenol-chloroform, gelpurification orkits commercially available on the market.

After endonuclease digestion the DNA may be gel-purified usingpolyacrylamide or agarose gel electrophoresis before ligation. The DNAcan be purified by standard molecular biology techniques (Sambrook etal. M: A Laboratory Manual, Sambrook et al. Molecular Cloning. ALaboratory Manual, Cold Spring Harbor, Laboratory Press, 1989) orapplying commercially available kits such as, but not limited to,QIAquick gel extraction kit (Qiagen, Inc., Chatsworth, Calif.).

Prior to the ligation reaction linearized vector fragments may betreated with bacterial alkaline phosphatase or calf intestine alkalinephosphatase to prevent self-ligation during the ligation step. Theligation reaction is preferably catalyzed by T4 DNA ligase. As known tothe routine practitioner ligation conditions can vary in time,temperature, concentration of buffers, quantities of DNA molecules to beligated, and amounts of ligase and ATP.

“Oligonucleotides” are short length, single- or double-strandedpolydeoxy nucleotides that are chemically synthesized by known methods.Alternatively, if the target amino acid sequence is known, one may inferpotential nucleic acid sequences using known and preferred codingresidues for each amino acid residue.

By “binding partner complex” is meant the association of two or moremolecules which are bound to each other in a specific, detectablemanner, thus the association of ligand and receptor, antibody andantigen.

The synthetic process for obtaining the compounds of the invention canadvantageously be carried out as parallel array synthesis to yieldlibraries of template-fixed ρ-hairpin mimetics of the above generalformula I. Such parallel synthesis allows one to obtain arrays ofnumerous (normally 24 to 192, typically 96) compounds of general formulaI in high yields and defined purities, minimizing the formation ofdimeric and polymeric by-products. The proper choice of thefunctionalized solid-support (i.e. solid support plus linker molecule),templates and site of cyclization play thereby key roles.

The functionalized solid support is conveniently derived frompolystyrene crosslinked with, preferably 1-5%, divinylbenzene;polystyrene grafted with polyethyleneglycol spacers (Tentagel^(R)); andpolyacrylamide resins (see also Obrecht, D.; Villalgordo, J.-M,“Solid-Supported Combinatorial and Parallel Synthesis ofSmall-Molecular-Weight Compound Libraries”, Tetrahedron OrganicChemistry Series, Vol. 17, Pergamon, Elsevier Science, 1998).

The solid support is functionalized by means of a linker, i.e. abifunctional spacer molecule which contains on one end an anchoringgroup for attachment to the solid support and on the other end aselectively cleavable functional group used for the subsequent chemicaltransformations and cleavage procedures. For the purposes of the presentinvention the linker must be designed to eventually release the carboxylgroup under mild acidic conditions which do not affect protecting groupspresent on any functional group in the side-chains of the various aminoacids. Linkers which are suitable for the purposes of the presentinvention form acid-labile esters with the carboxyl group of the aminoacids, usually acid-labile benzyl, benzhydryl and trityl esters;examples of linker structures of this kind include2-methoxy-4-hydroxymethylphenoxy (Sasrin^(R) linker),4-(2,4-dimethoxyphenyl-hydroxymethyl)-phenoxy (Rink linker),4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB linker), trityland 2-chlorotrityl.

Preferably, the support is derived from polystyrene crosslinked with,most preferably, 1-5%, divinylbenzene and functionalized by means of the2-chlorotrityl linker.

When carried out as a parallel array synthesis the process of theinvention can be advantageously carried out as described herein belowbut it will be immediately apparent to those skilled in the art how thisprocedure will have to be modified in case it is desired to synthesizeone single compound of the above formula I.

A number of reaction vessels (normally 24 to 192, typically 96) equal tothe total number of compounds to be synthesized by the parallel methodare loaded with 25 to 1000 mg, preferably 100 mg, of the appropriatefunctionalized solid support, preferably 1 to 3% cross linkedpolystyrene or tentagel resin.

The solvent to be used must be capable of swelling the resin andincludes, but is not limited to, dichloromethane (DCM),dimethylformamide (DMF), N-methylpyrrolidone (NMP), dioxane, toluene,tetrahydrofuran (THF), ethanol (EtOH), trifluoroethanol (TFE),isopropylalcohol and the like. Solvent mixtures containing at least onecomponent of a polar solvent (e.g. 20% TFE/DCM, 35% THF/NMP) arebeneficial for ensuring high reactivity and solvation of the resin-boundpeptide chains (Fields, G. B., Fields, C. G., J. Am. Chem. Soc. 1991,113, 4202-4207).

With the development of various linkers that release the C-terminalcarboxylic acid group under mild acidic conditions, not affectingacid-labile groups protecting functional groups in the side chain(s),considerable progresses have been made in the synthesis of protectedpeptide fragments. The 2-methoxy-4-hydroxybenzylalcohol-derived linker(Sasrin^(R) linker, Mergler et al., Tetrahedron Lett. 1988, 294005-4008) is cleavable with diluted trifluoroacetic acid (0.5-1% TFA inDCM) and is stable to Fmoc deprotection conditions during the peptidesynthesis, Boc/tBu-based additional protecting groups being compatiblewith this protection scheme. Other linkers which are suitable for theprocess of the invention include the super acid labile4-(2,4-dimethoxyphenyl-hydroxymethyl)-phenoxy linker (Rink linker, Rink,H. Tetrahedron Lett. 1987, 28, 3787-3790), where the removal of thepeptide requires 10% acetic acid in DCM or 0.2% trifluoroacetic acid inDCM; the 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid-derived linker(HMPB-linker, Flörsheimer & Riniker, Peptides 1991, 1990 131) which isalso cleaved with 1% TFA/DCM in order to yield a peptide fragmentcontaining all acid labile side-chain protective groups; and, inaddition, the 2-chlorotritylchloride linker (Barlos et al., TetrahedronLett. 1989, 30, 3943-3946), which allows the peptide detachment using amixture of glacial acetic acid/trifluoroethanol/DCM (1:2:7) for 30 min.

Suitable protecting groups for amino acids and, respectively, for theirresidues are, for example,

-   -   for the amino group (as is present also in the side-chain of        lysine)    -   Cbz benzyloxycarbonyl    -   Boc tert.-butyloxycarbonyl    -   Fmoc 9-fluorenylmethoxycarbonyl    -   Alloc allyloxycarbonyl    -   Teoc trimethylsilylethoxycarbonyl    -   Tcc trichloroethoxycarbonyl    -   Nps o-nitrophenylsulfonyl;    -   Trt triphenymethyl (or trityl)    -   for the carboxyl group (as is present also in the side-chain of        aspartic and glutamic acid) by conversion into esters with the        alcohol components    -   tBu tert.-butyl    -   Bn benzyl    -   Me methyl    -   Ph phenyl    -   Pac Phenacyl    -   Allyl    -   Tse trimethylsilylethyl    -   Tce trichloroethyl    -   for the guanidino group (as is present in the side-chain of        arginine)    -   Boc t-Butyloxycarbonyl    -   Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl    -   Ts tosyl (i.e. p-toluenesulfonyl)    -   Cbz benzyloxycarbonyl    -   Pbf pentamethyldihydrobenzofuran-5-sulfonyl    -   for the hydroxy group (as is present e.g. in the side-chain of        threonine and serine)    -   tBu tert.-butyl    -   Bn benzyl    -   Trt trityl    -   and for the mercapto group (as is present in the side-chain of        cysteine)    -   Acm acetamidomethyl    -   tBu tert.-butyl    -   Bn benzyl    -   Trt trityl    -   Mtr 4-methoxytrityl.

The 9-fluorenylmethoxycarbonyl-(Fmoc)-protected amino acid derivativesare preferably used as the building blocks for the construction of thetemplate-fixed β-hairpin loop mimetics of formula I. For thedeprotection, i.e. cleaving off of the Fmoc group, 20% piperidine in DMFor 2% DBU/2% piperidine in DMF can be used.

The quantity of the reactant, i.e. of the amino acid derivative, isusually 1 to 20 equivalents based on the mmols per gram (meq/g) loadingof the functionalized solid support (typically 0.1 to 2.85 mmol/g forpolystyrene resins) originally weighed into the reaction tube.Additional equivalents of reactants can be used if required to drive thereaction to completion in a reasonable time. The reaction tubes, incombination with the holder block and the manifold, are reinserted intothe reservoir block and the apparatus is fastened together. Gas flowthrough the manifold is initiated to provide a controlled environment,for example, nitrogen, argon, air and the like. The gas flow may also beheated or chilled prior to flow through the manifold. Heating or coolingof the reaction wells is achieved by heating the reaction block orcooling externally with isopropanol/dry ice and the like to bring aboutthe desired synthetic reactions. Agitation is achieved by shaking ormagnetic stirring (within the reaction tube). The preferred workstations(without, however, being limited thereto) are ACT 90, Symphoni abi 433Apeptide synthesizer and MultiSyn Tech's-Syro synthesizer.

Amide bond formation requires the activation of the α-carboxyl group forthe acylation step. When this activation is being carried out by meansof the commonly used carbodiimides such as dicyclohexylcarbodiimide(DCC, Sheehan & Hess, J. Am. Chem. Soc. 1955, 77, 1067-1068) ordiisopropylcarbodiimide (DIC, Sarantakis et al Biochem. Biophys. Res.Commun. 1976, 73, 336-342), the resulting dicyclohexylurea anddiisopropylurea is insoluble and, respectively, is soluble in thesolvents generally used. In a variation of the carbodiimide method1-hydroxybenzotriazole (HOBt, Konig & Geiger, Chem. Ber 1970, 103,788-798) is included as an additive to the coupling mixture. HOBtprevents dehydration, suppresses racemization of the activated aminoacids and acts as a catalyst to improve the sluggish coupling reactions.Certain phosphonium reagents have been used as direct coupling reagents,such as benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP) (Castro et al., Tetrahedron Lett. 1975, 14,1219-1222; Synthesis, 1976, 751-752), orbenzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophoshate(Py-BOP, Coste et al., Tetrahedron Lett. 1990, 31, 205-208), or2-(1H-benzotriazol-1-yl-)1,1,3,3-tetramethyluronium terafluoroborate(TBTU), or hexafluorophosphate (HBTU, Knorr et al., Tetrahedron Lett.1989, 30, 1927-1930); these phosphonium reagents are also suitable forin situ formation of HOBt esters with the protected amino acidderivatives. More recently diphenoxyphosphoryl azide (DPPA) orO-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TATU) orO-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU)/7-aza-1-hydroxy benzotriazole (HOAt, Carpinoet al., Tetrahedron Lett. 1994, 35, 2279-2281) have also been used ascoupling reagents.

Due to the fact that near-quantitative coupling reactions are essential,it is desirable to have experimental evidence for completion of thereactions. The ninhydrin test (Kaiser et al., Anal. Biochemistry 1970,34, 595), where a positive colorimetric response to an aliquot ofresin-bound peptide indicates qualitatively the presence of the primaryamine, can easily and quickly be performed after each coupling step.Fmoc chemistry allows the spectrophotometric detection of the Fmocchromophore when it is released with the base (Meienhofer et al., Int.J. Peptide Protein Res. 1979, 13, 35-42).

The resin-bound intermediate within each reaction tube is washed free ofexcess of retained reagents, of solvents, and of by-products byrepetitive exposure to pure solvent(s) by one of the two followingmethods:

1) The reaction wells are filled with solvent (preferably 5 ml), thereaction tubes, in combination with the holder block and manifold, areimmersed and agitated for 5 to 300 minutes, preferably 15 minutes, anddrained by gravity followed by gas pressure applied through the manifoldinlet (while closing the outlet) to expel the solvent.

2) The manifold is removed from the holder block, aliquots of solvent(preferably 5 ml) are dispensed through the top of the reaction tubesand drained by gravity through a filter into a receiving vessel such asa test tube or vial.

Both of the above washing procedures are repeated up to about 50 times(preferably about 10 times), monitoring the efficiency of reagent,solvent, and byproduct removal by methods such as TLC, GC, or inspectionof the washings.

The above described procedure of reacting the resin-bound compound withreagents within the reaction wells followed by removal of excessreagents, by-products, and solvents is repeated with each successivetransformation until the final resin-bound fully protected linearpeptide has been obtained.

Detachment of the fully protected linear peptide from the solid supportis achieved by immersion of the reaction tubes, in combination with theholder block and manifold, in reaction wells containing a solution ofthe cleavage reagent (preferably 3 to 5 ml). Gas flow, temperaturecontrol, agitation, and reaction monitoring are implemented as describedabove and as desired to effect the detachment reaction. The reactiontubes, in combination with the holder block and manifold, aredisassembled from the reservoir block and raised above the solutionlevel but below the upper lip of the reaction wells, and gas pressure isapplied through the manifold inlet (while closing the outlet) toefficiently expel the final product solution into the reservoir wells.The resin remaining in the reaction tubes is then washed 2 to 5 times asabove with 3 to 5 ml of an appropriate solvent to extract (wash out) asmuch of the detached product as possible. The product solutions thusobtained are combined, taking care to avoid cross-mixing. The individualsolutions/extracts are then manipulated as needed to isolate the finalcompounds. Typical manipulations include, but are not limited to,evaporation, concentration, liquid/liquid extraction, acidification,basification, neutralization or additional reactions in solution.

The solutions containing fully protected linear peptide derivativeswhich have been cleaved off from the solid support and neutralized witha base, are evaporated. Before this fully protected linear peptide isdetached from the solid support, it is possible, if desired, toselectively deprotect the protected α-amino group of the N-terminalamino acid residue and to acylate the amino group thus liberated bymeans of an acylating agent corresponding to the acyl substituent to beintroduced. Alternatively the protecting groups of the cysteines can befirst selectively removed and cyclisation can be effected as describedbelow. The cleavage from the resin and the deprotection of the cyclicpeptide can be done as described below.

The fully protected peptide derivative is treated with 82.5% TFA, 5%H₂O, 5% phenol, 5% thioanisol, 2.5% ethanthiol or another combination ofscavengers for effecting the cleavage of protecting groups. The cleavagereaction time is commonly 30 minutes to 12 hours, preferably about 5hours. Thereafter most of the TFA is evaporated and the product isprecipitated with ether or other solvents which are suitable therefor.After careful removal of the solvent, the peptide derivative obtainedcan be purified.

Cyclization (formation of the disulfide bridge) is then effected insolution using solvents such as water, DMF and the like. Variousoxidation reagents can be used for the cyclization, such as H₂O₂, air,or iodine. The duration of the cyclization is about 15 minutes to 24hours, preferably about 40 minutes. The progress of the reaction isfollowed, e.g. by RP-HPLC (Reverse Phase High Performance LiquidChromatography) and mass spectrometry. Then the solvent is removed byevaporation, and the cyclic peptide derivative is purified by RP-HPLC.

The phage display process of the invention can be carried out asfollowed:

The template fixed β-hairpin loop mimetic of the invention is fused toat least a portion of phage coat protein to form a fusion proteincontaining the template fixed β-hairpin loop mimetic. The fusion proteincan be made by expressing a gene fusion encoding the fusion proteinusing known techniques of phage display such as those described below.

Bacteriophage phage display is a known technique by which variantpolypeptides are displayed as fusion proteins to the coat protein on thesurface of bacteriophage particles (Scott, J. K. and Smith, G. P.Science 1990, 249; 386). The utility lies in the fact that largelibraries of selectively randomized protein variants (or randomly clonedcDNAs) can be rapidly and efficiently sorted for those sequences thatbind to a target molecule with high affinity.

Typically, variant polypeptides such as the template fixed β-hairpinmimetic of the invention, are fused to gene III protein which isdisplayed at one end of the virion.

Monovalent phage display is a process in which a protein or peptidesequence is fused to a portion of a gene III protein and expressed atlow levels in the presence of wild type gene III protein so thatparticles display mostly wild-type gene III protein and one copy or noneof the fusion protein which can also be used within the invention.

Suitable gene III vectors for display of template fixed β-hairpinmimetic of the invention include fUSE5, MKE 13 (New England Biolabs,Inc), fAFF1 (Cwirla et al, Proc. Natl. Acad. Sci. USA, 1990, 87,6378-6382), fd-CAT1, fdtetDOG, 33, 88, pComb3, pComb8, m663, pHEN1,pCANTAB5E genentech vectors CB and the like.

Phage display methods for proteins, peptides and mutated variantsthereof, including constructing a family of variant replicable vectorscontaining a transcription regulatory element operably linked to a genefusion encoding a fusion polypeptide, transforming suitable host cells,culturing the transformed cells to form phage particles which displaythe fusion polypeptide on the surface of the phage particle contactingthe recombinant phage particles with a target molecule so that at leasta portion of the particle bind to the target, and separating theparticles which bind from those that do not bind, are known and maybeused in accordance with the invention (O'Neil K. and Hoess R., Curr.Opin Struct. Biol. 1995 5, 443-449).

The gene encoding the coat protein of the phage and the gene encodingthe desired template fixed β-hairpin mimetic portion of the fusionprotein can be obtained by methods known in the art (Sambrook et al.Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, LaboratoryPress, pp. A1-A4 1989). The DNA encoding the gene may be chemicallysynthesized (Letzinger and Khorona. J. Am. Chem. Soc. 1965, 87, 3526,ibd. 1966, 88, 3181; L. J. McBride, M. H. Caruthers, Tetrahedron Lett.1983, 24, 245-248), and then mutated to prepare a library of variants asdescribed below.

To ligate DNA fragments together to form a functional vector containingthe fusion gene, the ends of the DNA fragments must be compatible witheach other. It may be necessary to first convert the sticky endscommonly produced by endonuclease digestion to blunt ends to make themcompatible for ligation. To blunt the ends, the DNA is treated in asuitable buffer for at least 15 minutes at 15° C. of the Klenow fragmentof DNA polymerase 1 (Klenow) in the presence of the four deoxynucleotidetriphosphates. The DNA is then purified by phenol-chloroform extractionand ethanol precipitation or other DNA purification technique.

The cleaved DNA fragments may be size separated and selected using gelelectrophoresis. The DNA may be electrophoresed through either anagarose or a polyacrylamide matrix. After electrophoresis the DNA isextracted from the matrix by electroelution or methods for purificationand ligation.

The DNA fragments that are to be ligated together are put in solution inabout equimolar amounts. The solution will also contain ATP, ligasebuffer and a ligase such as T4 DNA ligase.

After ligation the vector with the foreign gene now inserted is purifiedby standard molecular biology methods (Sambrook et al. MolecularCloning. A Laboratory Manual, Cold Spring Harbor, Laboratory Press,1989) and transformed into a suitable host cell. The preferred method oftransformation is electroporation which may be carried out using methodsknown in the art. For library construction, the DNA is preferablypresent at a final concentration of 0.05-0.2 microgram per 100microliter of competent cells suspension.

The DNA is preferably purified to remove contaminants. The DNA may bepurified by any known method; however, a preferred purification methodis the use of DNA affinity purification. The purification of DNA usingDNA binding resins and affinity reagents is well known and any of themethods well known in the art (eg. Biorad, Qiagene) can be used in thisinvention.

Any suitable cells which can be transformed by electroporation may beused as host cells in the method of the invention. Suitable host cellswhich can be transformed include gram negative cells such as E. coli.Suitable E. coli strains include, but are not limited to, XL1 Blue(Stratagene), ElectroTen-Blue (Stratagene,), ER2738 (New EnglandBiolabs), DH5α (Gibco), MC1061 (American Type Culture Collection (ATTC),ATTC number 53338).

Cell concentration of about 10¹⁰ colony forming units per ml ofsuspension of viable living cells and greater are preferably used duringelectroporation. After electroporation, cells are preferably grown inSOC medium. (for preparation of SOC medium see, for example, Sambrook etal. Molecular Cloning. A Laboratory Manual, Cold Spring Harbor,Laboratory Press, pp. A1-A4 1989).

If the amino acids are located close together in the polypeptide chain,they may be mutated simultaneously using one oligonucleotide thatencodes for all of the desired amino acid substitutions.Oligonucleotides can be synthesized which contain ambiguous orunambiguous nucleotides at predefined positions such as encoding thetemplates of the invention. At the ambiguous positions a mixture of allnucleotides or a selected subset of the nucleotides are included duringthe synthesis. Codons encoding the complete collection of the aminoacids can be realized, for example, by the NNK or NNS codon, where N isA, C, G, or T, and K is G or T and S is G or C.

After selection of the transformed cells, these cells are grown inculture and the vector DNA may then be isolated. Phage or phagemidvector DNA can be isolated, purified and analysed by DNA sequencingusing methods known in the art.

The present invention demonstrates the advantage of a novel system forrationally designing and analyzing peptides of well-defined structuralfeatures. The combinatorial libraries comprising such template fixedβ-hairpin mimetics and methods of using thereof provide usefulinformation and tools for exploring protein-protein interaction. Thetemplate fixed β-hairpin mimetics disclosed herein or generatedaccording to the disclosure of the invention can be candidates forvarious biological or therapeutic agents, including but not limited toenzyme inhibitors, ligand antagonists or ligand agonists.

The following Examples illustrate the invention in more detail but arenot intended to limit its scope in any way. The following abbreviationsare used in these Examples:

-   HBTU: 1-benzotriazol-1-yl-tetramethylurounium hexafluorophosphate    (Knorr et al. Tetrahedron Lett. 1989, 30, 1927-1930)-   HOBt: 1-hydroxybenzotriazole-   DIEA: diisopropylethylamine

EXAMPLES 1. Peptide Synthesis of Template Constrained β-Hairpin Mimetics

a) Synthesis

Coupling of the First Protected Amino Acid Residue

0.5 g of 2-chlorotritylchloride resin (Barlos et al. Tetrahedron Lett.1989, 30, 3943-3946) (0.83 mmol/g, 0.415 mmol) was filled into a driedflask. The resin was suspended in CH₂Cl₂ (2.5 ml) and allowed to swellat room temperature under constant stirring for 30 min. The resin wastreated with 0.415 mMol (1 eq) of the first suitably protected aminoacid residue (see below) and 284 μl (4 eq) of diisopropylethylamine(DIEA) in CH₂Cl₂ (2.5 ml), and the mixture was shaken at 25° C. for 4hours. The resin colour changed to purple and the solution remainedyellowish. The resin was shaken (30 ml of CH₂Cl₂/MeOH/DIEA:17/2/1) for30 min, then washed in the following order with CH₂Cl₂ (1×), DMF (1×),CH₂Cl₂ (1×), MeOH (1×), CH₂Cl₂ (1×), MeOH (1×), CH₂Cl₂ (2×), Et₂O (2×)and dried under vacuum for 6 hours.

Loading was typically 0.45-0.5 mMol/g.

The following preloaded resins were prepared:Fmoc-Cys(Trt)-chlorotritylresin, Fmoc-Glu(OtBu)-chlorotritylresin,Fmoc-Lys(Boc)-chlorotritylresin, Fmoc-Val-chlorotritylresin andFmoc-Gly-chlorotritylresin.

Procedure 1

The synthesis was carried out using a Syro-peptide synthesizer(Multisyntech) using 24 to 96 reaction vessels. In each vessel wasplaced 60 mg (weight of the resin before loading) of the above resin.The following reaction cycles were programmed and carried out:

Step Reagent Time 1 CH₂Cl₂, wash and swell (manual) 3 × 1 min. 2 DMF,wash and swell 1 × 5 min  3 40% piperidine/DMF 1 × 5 min. 4 DMF, wash 5× 2 min. 5 5 equiv. Fmoc amino acid/DMF +5 eq. HBTU +5 eq. HOBt +5 eq.DIEA 1 × 120 min   6 DMF, wash 4 × 2 min. 7 CH₂Cl₂, wash (at the end ofthe synthesis) 3 × 2 min.

Steps 3 to 6 are repeated to add each amino-acid.

Acetylation of the Amino Terminal Amino Acid

Steps 1-4 of procedure 1 were carried out in order to remove the Fmocprotecting group from the N-terminus of the synthesized sequence.

The resins loaded with the peptides were then transferred into 15 mlsyringes equipped with a frit and a stopcock. The resins were swelledduring 30 minutes with 5 ml of CH₂Cl₂. DIEA (0.4 ml) and aceticanhydride (0.1 ml) were added to each reactor. The resins were shakenduring 6 hours to one night. The resins were filtered and washed withsuccessively CH₂Cl₂/MeOH/CH₂Cl₂/MeOH/CH₂Cl₂/diethylether. The resinswere dried under vacuum.

Cleavage and Deprotection of the Fully Protected Peptide Fragment

After completion of the synthesis, the resin was suspended in 1 ml of 1%TFA in CH₂Cl₂ (v/v) and 1 ml of 20% DIEA in CH₂Cl₂ for 3 minutes. Thisprocedure was repeated three times to ensure completion of the cleavage.The filtrate was evaporated to dryness and the product was fullydeprotected with the cleavage mixture containing 82.5% trifluoroaceticacid (TFA), 5% water, 5% phenol, 5% thioanisole, and 2.5% ethanedithiolfor 5 h at room temperature and then concentrated under vacuum. Thepeptides were precipitated by adding 10 ml of diethylether, thencentrifugated, and the ether phase was removed. The operation wasrepeated twice with 5 ml of diethylether.

The crude peptides were dissolved in 1 ml of 10% CH₃CN in water and 0.5to 1 ml of DMF, filtered on celite and purified by preparative reversephase HPLC.

Cyclisation of the Linear Deprotected Peptide

The linear peptides obtained were dissolved in 1.5 ml of water at aconcentration of 10⁻⁴M and 15 μl of H₂O₂ (0.01M, 1 eq.) were added. Thecyclisation time was up to 700 min.

The obtained cyclic peptides were analysed by analytical HPLC andESI-MS. The analytical data comprising HPLC retention times and ESI-MSare indicated in the examples.

Analytical HPLC retention times (RT, in minutes) were determined using aVYDAC 218MS5215 column of size 0.21 cm×15 cm, 5 μm packing side (silica)with the following solvents A (H₂O+0.02% TFA) and B (CH₃CN) and thefollowing gradient: 0 min: 92% A, 8% B; 8 min: 62% A 38% B; 9-12 min: 0%A, 100% B, flow: 0.4 ml/min.

Example 1 (n=8) is shown in table 1. The peptide was synthesizedstarting with the amino acid Cys which was grafted to the resin.Starting resin was Fmoc-Cys(Trt)-chlorotritylresin which was prepared asdescribed above. The linear peptide was synthesized on solid supportaccording to procedure 1 in the following sequence:Resin-Cys-P8-P7-P6-P5-P4-P3-P2-P1-Cys, and it was then acylated,cleaved, deprotected, purified and cyclized, as indicated.HPLC-retention time (minutes) and mass were determined using thegradient described above: RT=7.26 min, [M+H]⁺=1281.3.

Example 2 (n=8) is shown in table 1. The peptide was synthesizedstarting with the amino acid Lys which was grafted to the resin.Starting resin was Fmoc-Lys(Boc)-chlorotritylresin which was prepared asdescribed above. The linear peptide was synthesized on solid supportaccording to procedure 1 in the following sequence:Resin-R²-Cys-P8-P7-P6-P5-P4-P3-P2-P1-Cys-R¹, and it was then acylated,cleaved, deprotected, purified and cyclized, as indicated.HPLC-retention time (minutes) and mass were determined using thegradient described above: RT=6.41 min, [M+H]⁺=871.4.

Example 3 (n=10) is shown in table 2. The peptide was synthesizedstarting with the amino acid Cys which was grafted to the resin.Starting resin was Fmoc-Cys(Trt)-chlorotritylresin which was prepared asdescribed above. The linear peptide was synthesized on solid supportaccording to procedure 1 in the following sequence:Resin-Cys-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1-Cys, and it was then acylated,cleaved, deprotected, purified and cyclized, as indicated.HPLC-retention time (minutes) and mass were determined using thegradient described above: RT=5.74 min, [M+H]⁺=779.2

Example 4 (n=10) is shown in table 2. The peptide was synthesizedstarting with the amino acid Lys which was grafted to the resin.Starting resin was Fmoc-Lys(Boc)-chlorotritylresin which was prepared asdescribed above. The linear peptide was synthesized on solid supportaccording to procedure 1 in the following sequence:Resin-R²-Cys-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1-Cys-R¹, and it was thenacylated, cleaved, deprotected, purified and cyclized, as indicated.HPLC-retention time (minutes) and mass were determined using thegradient described above: RT=5.13 min, [M+H]⁺=1009.2.

Example 5 (n=10) is shown in table 2. The peptide was synthesizedstarting with the amino acid Cys which was grafted to the resin.Starting resins was Fmoc-Cys(Trt)-chlorotritylresin which was preparedas described above. The linear peptide was synthesized on solid supportaccording to procedure 1 in the following sequence:Resin-Cys-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1-Cys, and it was then acylated,cleaved, deprotected, purified and cyclized, as indicated.HPLC-retention time (minutes) and mass were determined using thegradient described above: RT=6.81 min, [M+H]⁺=1482.6.

Examples 6 and 7 (n=10) are shown in table 2. The peptides weresynthesized starting with the amino acid Val which was grafted to theresin. Starting resin was Fmoc-Val-chlorotritylresin which was preparedas described above. The linear peptides were synthesized on solidsupport according to procedure 1 in the following sequence:Resin-R²-Cys-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1-Cys-R¹, and they were thenacylated, cleaved, deprotected, purified and cyclized, as indicated.HPLC-retention time (minutes) and mass were determined using thegradient described above: Example 6: RT=7.24 min, [M+H]⁺=−977.0; Example7: RT=6.24 min, [M+H]⁺=941.2.

Example 8 (n=10) is shown in table 2. The peptide was synthesizedstarting with the amino acid Gly which was grafted to the resin.Starting resin was Fmoc-Gly-chlorotritylresin which was prepared asdescribed above. The linear peptide was synthesized on solid supportaccording to procedure 1 in the following sequence:Resin-R²-Cys-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1-Cys-R¹, and it was thenacylated, cleaved, deprotected, purified and cyclized, as indicated.HPLC-retention time (minutes) and mass were determined using thegradient described above: RT=6.39 min, [M+H]⁺=856.0.

Example 9 (n=10) is shown in table 2. The peptide was synthesizedstarting with the amino acid Lys which was grafted to the resin.Starting resin was Fmoc-Lys(Boc)-chlorotritylresin which was prepared asdescribed above. The linear peptide was synthesized on solid supportaccording to procedure 1 in the following sequence:Resin-R²-Cys-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1-Cys-R¹, and it was thenacylated, cleaved, deprotected, purified and cyclized, as indicated.HPLC-retention time (minutes) and mass were determined using thegradient described above: RT=6.18 min, [M+H]⁺=972.3.

Example 10 (n=10) is shown in table 3. The peptide was synthesizedstarting with the amino acid Lys which was grafted to the resin.Starting resin was Fmoc-Lys(Boc)-chlorotritylresin which was prepared asdescribed above. The linear peptide was synthesized on solid supportaccording to procedure 1 in the following sequence:Resin-R²-Cys-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1-Cys-R¹, and it was thenacylated, cleaved, deprotected, purified and cyclized, as indicated.HPLC-retention time (minutes) and mass were determined using thegradient described above: RT=5.49 min, [M+H]⁺=1142.0.

Example 11 (n=10) is shown in table 3. The peptide was synthesizedstarting with the amino acid Glu which was grafted to the resin.Starting resin was Fmoc-Glu(Boc)-chlorotritylresin which was prepared asdescribed above. The linear peptide was synthesized on solid supportaccording to procedure 1 in the following sequence:Resin-R²-Cys-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1-Cys-R¹, and it was thenacylated, cleaved, deprotected, purified and cyclized, as indicated.HPLC-retention time (minutes) and mass were determined using thegradient described above: RT=6.85 min, [M+H]⁺=1033.8.

Example 12 (n=10) is shown in table 3. The peptide was synthesizedstarting with the amino acid Gly which was grafted to the resin.Starting resin was Fmoc-Gly-chlorotritylresin which was prepared asdescribed above. The linear peptide was synthesized on solid supportaccording to procedure 1 in the following sequence:Resin-R²-Cys-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1-Cys-R¹, and it was thenacylated, cleaved, deprotected, purified and cyclized, as indicated.HPLC-retention time (minutes) and mass were determined using thegradient described above: RT=6.41 min, [M+H]⁺=913.0.

Example 13 (n=12) is shown in table 4. The peptide was synthesizedstarting with the amino acid Cys which was grafted to the resin.Starting resin was Fmoc-Cys(Trt)-chlorotritylresin which was prepared asdescribed above. The linear peptide was synthesized on solid supportaccording to procedure 1 in the following sequence:Resin-Cys-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1-Cys, and it was thenacylated, cleaved, deprotected, purified and cyclized, as indicated.HPLC-retention time (minutes) and mass were determined using thegradient described above: RT=5.74 min, [M+H]⁺=836.5.

Example 14 (n=12) is shown in table 4. The peptide was synthesizedstarting with the amino acid Lys which was grafted to the resin.Starting resin was Fmoc-Lys(Boc)-chlorotritylresin which was prepared asdescribed above. The linear peptide was synthesized on solid supportaccording to procedure 1 in the following sequence:Resin-R²-Cys-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1-Cys-R¹, and it wasthen acylated, cleaved, deprotected, purified and cyclized, asindicated. HPLC-retention time (minutes) and mass were determined usingthe gradient described above: RT=5.01 min, [M+H]⁺=1065.1.

2a. Method of Measuring the Kinetics of Disulfide Bridge Formation ofTemplate-Fixed β-Hairpin Mimetics

Stock solutions of each linear deprotected, purified peptide wereprepared, containing 1.5 ml of the peptide solution at a concentrationof 10⁴M in water. The formation of the disulfide bridge was monitored onanalytical LC-MS as described above. The first data point is performedwithout the oxidation reagent H₂O₂ at time t 0. The second data point isperformed 15 minutes after adding the oxidation reagent H₂O₂ (15 μl,0.01M, 1 eq.). Data points were recorded every 33 minutes up to a timeafter which no progress of conversion was detected.

The amount of disulfide bridged cyclic peptide was calculated based onpeak area percentage (manually integrated) of the cyclic peptide at timet minus peak area percentage of the cyclic peptide (manually integrated)at time t0 at a wave length of 220 nm.

2b. Method of Measuring Circular Dichroism

Circular dichroism measurements are sensitive to the secondary structureof both peptides and proteins and have been extensively used to examinethe conformation of both (M. Jourdan, S. R. Griffiths-Jones, M. S.Searle, Eur. J. Biochem. 2000, 267, 3539-3548; J. T. Pelto, L. R. Mc.Lean, Analytical Biochemistry, 2000, 277, 167-176).

Circular Dichroism spectra were obtained on a Jasco J-715spectropolarimeter, equipped with a spectra manager for windows 95/NT.Version 1.52.01 [Build2]. All measurements were performed at roomtemperature in quartz cells of 0.1 cm path length in water. Spectra wererecorded with a 1 nm bandwidth, five scans were collected to improve thesignal-to-noise ratio and the solvent baseline was recorded andsubtracted from the spectra of the samples. All CD spectra were smoothed(with the same value), and are reported as molecular ellipticity units(Mol. Ellip.) of peptide residue.

Measurement parameters: Band width: 1.0 nm, response: 1 s, sensitivity:standard, measurement range: 240-190 nm, data pitch: 0.5 nm, scanningspeed: 50 nm/min Concentration of the linear deprotected, purifiedpeptide solutions: 10⁴M in water as TFA salts. The pH and purity of thelinear deprotected, purified peptides as precursors of the followingexamples were: Example 3: pH 6.38, purity 91%; Example 4: pH 6.77,purity 89%; Example 5: pH 6.01, purity 98%; Example 9: pH 6.00, purity94%.

2c. Results

FIGS. 1-6 show a comparison of the formation of disulfide bridgedβ-hairpin mimetics in % up to a time after which no progress ofconversion was detected.

FIG. 1: Examples 1 and 2 (n=8); disulfide bridge formation rate of thecompound of Example 2 having a template is compared to Example 1 as areference not having a template.

FIG. 2: Examples 3 and 4 (n=10); disulfide bridge formation rate of thecompound of Example 4 having a template is compared to Example 3 as areference not having a template.

FIG. 3: Examples 5-9 (n=10); disulfide bridge formation rates of thecompounds of Examples 6-9 having a template are compared to Example 5 asa reference not having a template.

FIG. 4: Examples 3 and 10 (n=10); disulfide bridge formation rate of thecompound of Example 10 having a template is compared to Example 3 as areference not having a template.

FIG. 5: Examples 5, 11 and 12 (n=10); disulfide bridge formation ratesof the compounds of Examples 11 and 12 having a template are compared toExample 5 as a reference not having a template.

FIG. 6: Example 13 and 14 (n=12); disulfide bridge formation rate of thecompound of Example 14 having a template is compared to Example 13 as areference not having a template.

FIG. 7: CD spectra (ε in deg×cm²/mol) of the linear peptide precursorsof the compounds of Examples 3, 4, 5 and 9, i.e. before disulfide bridgeformation.

2d. Discussion Design of the Example Sequences Z

Two different types of core peptide sequences Z have been chosen inorder to investigate whether the template is facilitating the formationand stabilization of template fixed β-hairpin mimetics: The sequence-Leu-Trp-Tyr-Ser-Asn-His-Trp-Val- [SEQ ID NO:22] was taken from the CDRL3 loop of an antibody (L. Jiang, K. Moehle, J. Robinson, Chimia (2000)54, 558-563) and was modified to the sequence-Lys-Trp-Phe-Ser-Asn-His-Tyr-Gln- [SEQ ID NO:23] containing astabilizing β-turn and a β-sheet sequence according to P. Y. Chou G. DFasman, J. Mol. Biol. (1977) 115, 135-175 as a reference β-hairpin. Asecond sequence Z -Phe-Leu-Ala-His-Tyr-Ala- [SEQ ID NO:24] wasconstructed from the sequence -Leu-Trp-Tyr-Ser-Asn-His-Trp-Val-Lys-Trp-[SEQ ID NO:25] which does not contain a dedicated stabilizing β-turnsequence or a β-sheet sequence according to P. Y. Chou G. D Fasman, J.Mol. Biol. (1977) 115, 135-175.

The results depicted in FIGS. 1-6 demonstrate that the disulfide bridgeformation rates of compounds with a template and the core sequence-Phe-Leu-Ala-His-Tyr-Ala- [SEQ ID NO:24] is faster compared to those ofcompounds containing the same core sequence but no template. The resultsdemonstrate clearly that the templates of the invention facilitate theformation of a β-hairpin mimetic effectively. Even in the case of thecore sequence Z -Lys-Trp-Phe-Ser-Asn-His-Tyr-Gln- [SEQ ID NO:23] whichis itself already containing a stabilizing β-turn and a β-sheetsequence, it can be demonstrated (see FIGS. 2 and 4) that the templatefacilitates the formation of a β-hairpin mimetic.

In addition the CD-spectrum (see FIG. 7) of the linear precursor of thecompound of Example 5 which is containing the core sequence-Phe-Leu-Ala-His-Tyr-Ala- [SEQ ID NO:24] but no template indicates ahigh content of coil- and α-helix structure and traces of a β-sheetstructure, whereas the linear precursor of the compound of Example 9(containing the same core sequence and a template) indicates a highcontent of β-sheet structure and a β-turn structure. The CD-spectrum ofthe linear precursor of the compound of Example 3 containing the coresequence Lys-Trp-Phe-Ser-Asn-His-Tyr-Gln- [SEQ ID NO:23] but no templateindicates a mixture of a coil structure and a β-sheet structure whereasthe CD spectrum of the linear precursor of the compound of Example 4(containing the same core structure and a template) indicates a highcontent of a β-sheet structure. These findings indicate that thetemplates of the invention induce the formation of a β-hairpin mimetic.

3. Construction of Phage Displayed Template Fixed β-Hairpin MimeticSequences Incorporating the Templates

Procedure 1:

The oligonucleotide libraries of the invention can be fused to the geneIII of the filamentous bacteriophage M13KE according to the procedure ofthe Ph. D. Peptide Display Cloning System, technical Bulletin #E8101(Aug. 21, 2002, New England Biolabs, Inc) and K. Noren, C. Noren,Methods, 2001, 23, 169-178.

Phage display of the template fixed hairpin mimetic of Example 3 [SEQ IDNO: 10] is accomplished as described in the following section. For allother sequences listed in tables 5-9 corresponding procedures are used,differing in oligonucleotides used to generate insert DNA.

Oligonucleotides 1 and 2 (see below) are used to construct insert DNA.Positions of the unique AccI and EagI restriction sites for cloning intovector DNA are underlined.

For annealing 2 μg (approx. 170 pmol) of oligonucleotide 1 and 4.5 μg ofoligonucleotide 2 (approx. 170 pmol) are heated to approximately 95° C.in 50 μl TE (10 mM Tris-HCL, pH 8.0, 1 mM EDTA) containing 100 mM NaCl.After slowly cooling down over 15-30 minutes the annealed duplex isextended with the Klenow fragment of DNA Polymerase I in a total volumeof 200 Reaction conditions are as outlined in Sambrook et al. MolecularCloning. A Laboratory Manual, Cold Spring Harbor, Laboratory Press. Theresulting insert DNA was digested with EagI and Acc65I using conditionsrecommended by the supplier (New England Biolabs). The mixture isextracted with phenol/chloroform and chloroform before precipitation ofthe aqueous phase with ethanol. The precipitate is purified on an 8%nondenaturing polyacrylamide gel using standard molecular biologyprocedures (Sambrook et al. Molecular Cloning. A Laboratory Manual, ColdSpring Harbor, Laboratory Press).

15 μg of M13KE vector (New England Biolabs) are digested with EagI andAcc65I according to the conditions recommended by New England Biolabs.The mixture is purified on agarose and linearized vector DNA isrecovered with the QIAquick gel extraction kit (Qiagen).

oligonucleotide number 1 [SEQ ID NO: 26]           Acc65I 5′CATGCCCGGGTACCTTTCTATTCTCACTCTGAAACCTGC 3′ oligonucleotide number 2[SEQ ID NO: 27]           EagI 5′CATGTTTCGGCCGAGCCACCACCTTTGGTGCAGGTCTGATAATGGTTGCTGAACCATTTGGTGCAGGTTTCAGAGTGAGAATAG′ 3′

For cloning of insert DNA ligation conditions are unoptimised.

Briefly, ligation is performed overnight at 16° C. in a total volume of20 μl T4 DNA ligase buffer containing approximately 40 ng linearizedvector, approximately 3:1 molar excess of duplex and 200 units of T4ligase. Control reactions containing vector only, plus and minus ligase,are also performed to determine ligation efficiencies and background dueto vector religation. The ligation mixtures are heat-inactivated at 65°C. for 15 minutes and 1 μl aliquots are used for subsequentelectroporation into 100 μl ElectroTen-blue electroporation competentcells (Stratagene) as outlined by New England Biolabs. Immediately afterelectroporation 1 ml SOC medium (2% Bacto tryptone, 0.5% Bacto yeastextract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) is added to each cuvette, and incubations are performed for 30minutes at 37° C. Aliquots thereof are used for titering each culture byblue/white selection using medium containing X-gal and IPTG. Individualclones for sequence confirmation are selected and incubated in 1 ml of1:100 dilutions of an overnight culture of XL1-Blue after incubation at37° C. for 4-4.5 hours. Phage for long-term storage and sequencingpurpose are obtained from these liquid cultures applying protocols wellknown in the art.

Procedure 2: (Randomized Template Fixed β-Hairpin Mimetic Libraries)

Phage display of the sequence of Example 15 [SEQ ID NO 42], see table 9,is representative for the preparation of randomized template fixedβ-hairpin mimetic libraries and is described in the following section.

Oligonucleotides 1 and 3 (see above and, respectively, below) are usedto construct insert DNA. Positions of the unique AccI and EagIrestriction sites for cloning into vector DNA are underlined.

For annealing 2 μg (approx. 170 pmol) of oligonucleotide 1 and 4.5 μg ofoligonucleotide 2 (approx. 170 pmol) are heated to approximately 95° C.in 50 μl TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) containing 100 mM NaCl.After slowly cooling down over 15-30 minutes the annealed duplex isextended with the Klenow fragment of DNA Polymerase I in a total volumeof 200 applying reaction conditions well known in the art (Sambrook etal. Molecular Cloning. A Laboratory Manual, Cold Spring Harbor,Laboratory Press). The resulting insert DNA is digested with EagI andAcc65I using conditions recommended by the New England Biolabs. Themixture is extracted with phenol/chloroform and chloroform beforeprecipitation of the aqueous phase with ethanol. The precipitate ispurified on an 8% non-denaturing polyacrylamide gel using standardmolecular biology procedures (Sambrook et al. Molecular Cloning. ALaboratory Manual, Cold Spring Harbor, Laboratory Press).

15 μg of M13KE vector New England Biolabs are digested with EagI andAcc651 according to the conditions recommended by the New EnglandBiolabs. The mixture is purified on agarose and linearized vector DNA isrecovered with the QIAquick gel extraction kit (Qiagen).

oligonucleotide number 1 [SEQ ID NO: 26]           Acc65I 5′CATGCCCGGGTACCTTTCTATTCTCACTCTGAAACCTGC 3′ oligonucleotide number 3[SEQ ID NO: 44]           EagI 5′CATGTTTCGGCCGAGCCACCACCTTTGGTGCAMNNMNNMNNMNNGTCACCACGMNNMNNMNNGCAGGTTTCAGAGTGAGAATAG 3′

Optimal ligation conditions are determined in a total volume of 20 μlvarying the molar ratio of insert:vector from 3:1, 5:1 to 10:1 and using40 and 100 ng of digested vector, respectively. Control reactionscontaining just vector, in the presence and absence of ligase, are alsoperformed to determine ligation efficiencies and background due tovector religation. Reactions are carried out overnight at 16° C. in 1×ligation buffer and 200 NEB units (=3 Weiss units) of T4 DNA ligase.Test ligations are heat-inactivated at 65° C. for 15 minutes. Subsequentelectroporations of 1 μl aliqouts into 100 μl ElectroTen-blueelectroporation competent cells (Stratagene) are performed according torecommendations outlined by the manufacturer. Immediately afterelectroporation 1 ml SOC medium (2% Bacto tryptone, 0.5% Bacto yeastextract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) are added to each cuvette. and incubations are performed for 30minutes at 37° C. Titers for plaque forming units of each outgrowthculture are determined by blue/white selection with X-gal followingstandard molecular biology procedures (Sambrook et al. MolecularCloning. A Laboratory Manual, Cold Spring Harbor, Laboratory Press).

Ligations displaying highest plaque/microgram input vector ratio arescaled up to obtain the desired library complexity. For libraryconstruction, the DNA is present at a final concentration ofapproximately 0.1 microgram per 100 microliter of competent cellssuspension. Immediately after electroporation 1 ml SOC medium is addedto each cuvette and the SOC outgrowths are grouped in pools of 5 andincubated for 30 minutes at 37° C. The library complexity is determinedby titering several outgrowths and the remainders are used for phageamplification. For amplification each pool of SOC outgrowths is added to1 liter of a 1:100 dilution of an overnight culture of XL1-Blue.Incubation is performed for 4.5-5 hours at 37° C. with vigorousaeration. Phage from these liquid cultures is obtained by clearing thesupernatant twice by centrifugation, and precipitating phage particleswith polyethylene glycol (final concentration 3.3% polyethyleneglycol-8000, 0.4 M NaCl) overnight at 4° C. After centrifugation theobtained pellet is redissolved in TBS, the suspension cleared bycentrifugation and phage particles are obtained from the supernatant byprecipitation with polyethylene glycol (as described above) for 1 hourat 4° C. The phage pellet after centrifugation is resuspended in TBS (50mM Tris-HCl, pH 7.5, 100 mM NaCl) and stored at 4° C.

TABLE 1 Examples 1-2, n = 8 Example Sequ.ID          R¹    Cys P1  P2  P3  P4  P5  P6  P7  P8  Cys  R² 1SEQ ID NO: 8           Ac-NH-Cys Lys Trp Phe Leu Ala His Tyr Ala Cys-H 2SEQ ID NO: 9Ac-NH-Glu Thr   Cys Lys Trp Phe Leu Ala His Tyr Ala Cys  Thr Lys-Hcysteines are linked by a disulfide bridge

TABLE 2 Examples 3-9, n = 10 Example Sequ.ID          R¹     Cys P1  P2  P3  P4  P5  P6   3 SEQ ID NO: 10          Ac-NH-Cys Thr Lys Trp Phe Ser Asn  4 SEQ ID NO: 11Ac-NH-Glu Thr   Cys Thr Lys Trp Phe Ser Asn  5 SEQ ID NO: 12          Ac-NH-Cys Thr Lys Trp Phe Leu Ala  6 SEQ ID NO: 13Ac-NH-Leu Glu   Cys Thr Lys Trp Phe Leu Ala  7 SEQ ID NO: 14Ac-NH-Asn Gly   Cys Thr Lys Trp Phe Leu Ala  8 SEQ ID NO: 15Ac-NH-Gly Gly   Cys Thr Lys Trp Phe Leu Ala  9 SEQ ID NO: 16Ac-NH-Glu Thr   Cys Thr Lys Trp Phe Leu Ala  ExampleP7  P8  P9  P10 Cys  R² 3 His Tyr Gln Thr Cys-H 4His Tyr Gln Thr Cys  Thr Lys-H 5 His Tyr Ala Thr Cys-H 6His Tyr Ala Thr Cys  Lys Val-H 7 His Tyr Ala Thr Cys  Lys Val-H 8His Tyr Ala Thr Cys  Gly Gly-H 9 His Tyr Ala Thr Cys  Thr Lys-Hcysteines are linked by a disulfide bridge Ac = Acetyl

TABLE 3 Examples 10-12, n = 10 Example Sequ.ID              R¹  Cys P1  P2  P3  P4  P5  P6    10 SEQ ID NO: 17Ac-NH-Glu Leu Lys Cys Thr Lys Trp Phe Ser Asn  11 SEQ ID NO  18Ac-NH-Lys Val Gly Cys Thr Lys Trp Phe Leu Ala 12 SEQ ID NO: 19Ac-NH-Gly Gly Gly Cys Thr Lys Trp Phe Leu Ala ExampleP7  P8  P9  P10 Cys R² 10 His Tyr Gln Thr Cys Glu Val Lys-H 11His Tyr Ala Thr Cys Gly Leu Glu-H 12 His Tyr Ala Thr Cys Gly Gly Gly-Hcysteines are linked by a disulfide bridge

TABLE 4 Examples 13-14, n = 12 Example Sequ.ID          R¹     Cys P1  P2  P3  P4  P5  P6   13 SEQ ID NO: 20           Ac-NH-Cys Gly Thr Lys Trp Phe Ser  14 SEQ ID NO: 21Ac-NH-Glu Thr   Cys Gly Thr Lys Trp Phe Ser  ExampleP7  P8  P9  P10 P11 P12 Cys  R² 13 Asn His Tyr Gln Thr Gly Cys-H 14Asn His Tyr Gln Thr Gly Cys  Thr Lys-H cysteines are linked by adisulfide bridge Ac = AcetylTable of DNA-Sequences Corresponding to Examples 1-14

TABLE 5 Examples 1-2, n = 8    R¹  Cys P1  P2  P3  P4  P5  P6  P7  P8  Cys R² SeqID No: 28        TGC AAA TGG TTC CTG GCG CAT TAT GCG TGC SeqID No: 29GAA ACC TGC AAA TGG TTC CTG GCG CAT TAT GCG TGC ACC AAA

TABLE 6 Examples 3-9, n = 10    R¹  Cys P1  P2  P3  P4  P5  P6  P7  P8  P9  P10 Cys R² SeqID No: 30        TGC ACC AAA TGG TTC AGC AAC CAT TAT CAG ACC TGC SeqID No: 31GAA ACC TGC ACC AAA TGG TTC AGC AAC CAT TAT CAG ACC TGC ACC AAASeqID No: 32         TGC ACC AAA TGG TTC CTG GCG CAT TAT GCG ACC TGCSeqID No: 33CTG GAA TGC ACC AAA TGG TTC CTG GCG CAT TAT GCG ACC TGC AAA GTTSeqID No: 34AAC GGT TGC ACC AAA TGG TTC CTG GCG CAT TAT GCG ACC TGC AAA GTTSeqID No: 35GGT GGT TGC ACC AAA TGG TTC CTG GCG CAT TAT GCG ACC TGC GGC GGTSeqID No: 36GAA ACC TGC ACC AAA TGG TTC CTG GCG CAT TAT GCG ACC TGC ACC AAA

TABLE 7 Examples 10-12 n = 10        R¹  Cys P1  P2  P3  P4  P5  P6  P7  P8  P9  P10 Cys R²SeqID No: 37GAA CTG AAA TGC ACC AAA TGG TTC AGC AAC CAT TAT CAG ACC TGC GAA GTT AAASeqID No: 38AAA GTT GGT TGC ACC AAA TGG TTC CTG GCG CAT TAT GCG ACC TGC GGT CTG GAASeqID No: 39GGT GGT GGC TGC ACC AAA TGG TTC CTG GCG CAT TAT GCG ACC TGC GGC GGT GGT

TABLE 8 Examples 13-14, n = 12 Seq. ID    R¹  Cys P1  P2  P3  P4  P5  P6  P7  P8  P9  P10 P11 P12 Cys R²SeqID No 40        TGC GGT ACC AAA TGG TTC AGC AAC CAT TAT CAG ACC GGT TGCSeqID No 41GAA ACC TGC GGT ACC AAA TGG TTC AGC AAC CAT TAT CAG ACC GGT TGC ACC AAA

TABLE 9 Example 15, n = 10, DNA Sequence and translated peptide sequenceof a randomized template fixed β-hairpin mimetic Phage library ExampleSeq. ID     R¹  Cys P1  P2  P3  P4  P5  P6  P7  P8  P9  P10 Cys R²Ex.15  SeqID No: 42GAA ACC TGC NNK NNK NNK CGT GGT GAC NNK NNK NNK NNK TGC ACC AAASeqID No: 43Glu Thr Cys X   X   X   Arg Gly Asp X   X   X   X   Cys Thr Lys X:randomized amino acid positions Cysteines are linked by a disulfidebridge

The invention claimed is:
 1. A library comprising a plurality ofcompounds of the formula R¹-Cys-Z-Cys-R² (I), in which each of the twoCys is the residue of L-cysteine, the two -SH groups in the twoL-cysteine residues being replaced by one -S-S- group, R¹ and R²comprise Glu-Thr and Thr-Lys; or Lys-Thr and Thr-Glu; or Thr-Glu andLys-Thr; or Thr-Lys and Glu-Thr; or Leu-Glu and Lys-Val; or Val-Lys andGlu-Leu; or Glu-Leu and Val-Lys; or Lys-Leu and Val-Glu; or Glu-Leu-Lysand Glu-Val-Lys; or Lys-Val-Glu and Lys-Leu-Glu; or Leu-Glu-Lys andGlu-Lys-Val; or Val-Lys-Glu and Lys-Glu-Leu; or Glu-Lys-Leu andVal-Glu-Lys; or Lys-Glu-Val and Leu-Lys-Glu; or Lys-Glu-Leu andVal-Lys-Glu; or Glu-Lys-Val and Leu-Glu-Lys, and Z is a chain of from 6up to and including 12 amino acid residues, each of these residuesbeing, independently, the residue of a naturally occurring L-alpha-aminoacid, with the proviso, that Z comprises one of the moieties-Lys-Trp-Phe-Ser-Asn-His-Tyr-Gln- [SEQ ID NO:23]; and-Phe-Leu-Ala-His-Tyr-Ala- [SEQ ID NO:24].
 2. A library according toclaim 1, characterized in that R¹ and R² are Glu-Thr and Thr-Lys; orLeu-Glu and Lys-Val; or Glu-Leu-Lys and Glu-Val-Lys.
 3. A libraryaccording to claim 1, wherein said compounds in said library arepeptides having a sequence selected from the group consisting of SEQ IDNO: 9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, and SEQID NO:21.
 4. A library according to claim 1, wherein each of thecompounds of the formula I is fused to at least a portion of a phagecoat protein and is displayed on the surface of a phage or phagemid. 5.A library according to claim 2, wherein each of the compounds of theformula I is fused to at least a portion of a phage coat protein and isdisplayed on the surface of a phage or phagemid.
 6. A library accordingto claim 3, wherein each of the compounds is fused to at least a portionof a phage coat protein and is displayed on the surface of a phage orphagemid.